Recombinant lysosomal enzymes; MPS IIID; MPS VI; caprine N-acetylglucosamine- 6-sulfatase; human N-acetylgalactosamine-4-sulfatase; lysosomal storage diseases.

1998 ◽  
Vol 11 (3) ◽  
pp. 233-242 ◽  
Author(s):  
N. Y. Walton ◽  
A. K. Nagy ◽  
D. M. Treiman
Keyword(s):  
Lysosomal Enzymes ◽  
Lysosomal Storage Diseases ◽  
Lysosomal Storage ◽  
Storage Diseases ◽  
Mps Vi ◽  
Mps Iiid

Development and Validation of Leukocyte Enzyme Activity Assay by LC-MS/MS for Diagnosis of Krabbe Disease and Other Lysosomal Storage Diseases

2020 ◽  
Vol 154 (Supplement_1) ◽  
pp. S16-S16
Author(s):  
Hsuan-Chieh Liao ◽  
Laura Mitchell ◽  
Katerina Sadilkova ◽  
Jane Dickerson ◽  
Rhona Jack ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Liquid Chromatography ◽  
Lysosomal Enzymes ◽  
Internal Standard ◽  
Lysosomal Storage Diseases ◽  
Krabbe Disease ◽  
Tandem Mass ◽  
Lysosomal Storage ◽  
Mass Detection ◽  
Storage Diseases

Abstract Background Deficiency of the lysosomal enzyme galactosylcerebrosidase (GALC) causes Krabbe disease. The diagnosis for Krabbe disease includes measurement of GALC enzymatic activity by radioisotope assay or accumulation of metabolite psychosine. To improve current diagnostic workflow and assay performance, we developed and validated a leukocyte enzymatic assay by using liquid chromatography tandem mass spectrometry (LC-MS/MS) for lysosomal storage diseases. Methods Leukocytes were separated and extracted from whole blood samples, and total protein was quantitated by BCA method. Commercialized and multiplexed substrates, internal standards, and buffer were incubated with cell lysates. The lysosomal enzymes in leukocytes metabolized the artificial substrate into product which is structurally identical to the internal standard. Liquid-liquid extraction was performed and supernatant was dried down and reconstituted. Liquid chromatography separation was achieved by Waters CSH C18, 2.1 x 50 mm column and Acquity UPLC system. A Waters Xevo TQS tandem mass spectrometer was used for mass detection. Results Enzymatic reaction products for six lysosomal enzymes were chromatographically resolved from substrate breakdown products through 3.5 minutes gradient liquid chromatography. Intra-assay imprecision was determined by 11 replicates of samples containing low and high concentration (CV<15%). Carryover was determined by assaying triplicates of cell lysate-free cocktails directly after injection of high enzyme activity sample (less than 0.1%). No matrix effect was found. The GALC enzyme activity was calculated and standardized by corresponding product and internal standard ratios from 5-point standard curve. The range of enzyme activity from three, known affected patients is 0.01–0.07 (nmol/hr/mg protein); whereas, two identified carriers had enzyme activate in the range of 0.14–0.40 (nmol/hr/mg protein). The reference interval was established from 63 residual, unaffected samples and was 0.12–5.97 (1.44±1.44) nmol/hr/mg protein. Conclusions A simple and multiplexed LC-MS/MS assay was developed which can measure small amounts of residual GALC enzyme activity in leukocytes. This confirmatory assay will aid in the diagnosis and prognosis (i.e. differentiate disease severity) of Krabbe disease and other lysosomal storage disorders.

scholarly journals Tandem Mass Spectrometry Has a Larger Analytical Range than Fluorescence Assays of Lysosomal Enzymes: Application to Newborn Screening and Diagnosis of Mucopolysaccharidoses Types II, IVA, and VI

2015 ◽  
Vol 61 (11) ◽  
pp. 1363-1371 ◽  
Author(s):  
Arun Babu Kumar ◽  
Sophia Masi ◽  
Farideh Ghomashchi ◽  
Naveen Kumar Chennamaneni ◽  
Makoto Ito ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Newborn Screening ◽  
Lysosomal Enzymes ◽  
Lysosomal Storage Diseases ◽  
Tandem Mass ◽  
Lysosomal Storage ◽  
Storage Diseases ◽  
Mps Ii ◽  
Analytical Range ◽  
Screening And Diagnosis

Abstract BACKGROUND There is interest in newborn screening and diagnosis of lysosomal storage diseases because of the development of treatment options that improve clinical outcome. Assays of lysosomal enzymes with high analytical range (ratio of assay response from the enzymatic reaction divided by the assay response due to nonenzymatic processes) are desirable because they are predicted to lead to a lower rate of false positives in population screening and to more accurate diagnoses. METHODS We designed new tandem mass spectrometry (MS/MS) assays that give the largest analytical ranges reported to date for the use of dried blood spots (DBS) for detection of mucopolysaccharidoses type II (MPS-II), MPS-IVA, and MPS-VI. For comparison, we carried out fluorometric assays of 6 lysosomal enzymes using 4-methylumbelliferyl (4MU)-substrate conjugates. RESULTS The MS/MS assays for MPS-II, -IVA, and -VI displayed analytical ranges that are 1–2 orders of magnitude higher than those for the corresponding fluorometric assays. The relatively small analytical ranges of the 4MU assays are due to the intrinsic fluorescence of the 4MU substrates, which cause high background in the assay response. CONCLUSIONS These highly reproducible MS/MS assays for MPS-II, -IVA, and -VI can support multiplex newborn screening of these lysosomal storage diseases. MS/MS assays of lysosomal enzymes outperform 4MU fluorometric assays in terms of analytical range. Ongoing pilot studies will allow us to gauge the impact of the increased analytical range on newborn screening performance.

Electron Microscopy in the diagnosis of lysosomal storage diseases

1989 ◽  
Vol 47 ◽  
pp. 866-867
Author(s):  
Carole Vogler ◽  
Harvey S. Rosenberg
Keyword(s):  
Electron Microscopy ◽  
Lysosomal Enzyme ◽  
Rectal Mucosa ◽  
Lysosomal Storage Diseases ◽  
Cell Types ◽  
Lysosomal Storage ◽  
Storage Material ◽  
Ultrastructural Examination ◽  
Blood Leukocytes ◽  
Storage Diseases

Diagnostic procedures for evaluation of patients with lysosomal storage diseases (LSD) seek to identify a deficiency of a responsible lysosomal enzyme or accumulation of a substance that requires the missing enzyme for degradation. Most patients with LSD have progressive neurological degeneration and may have a variety of musculoskeletal and visceral abnormalities. In the LSD, the abnormally diminished lysosomal enzyme results in accumulation of unmetabolized catabolites in distended lysosomes. Because of the subcellular morphology and size of lysosomes, electron microscopy is an ideal tool to study tissue from patients with suspected LSD. In patients with LSD all cells lack the specific lysosomal enzyme but the distribution of storage material is dependent on the extent of catabolism of the substrate in each cell type under normal circumstances. Lysosmal storages diseases affect many cell types and tissues. Storage material though does not accumulate in all tissues and cell types and may be different biochemically and morphologically in different tissues.Conjunctiva, skin, rectal mucosa and peripheral blood leukocytes may show ultrastructural evidence of lysosomal storage even in the absence of clinical findings and thus any of these tissues can be used for ultrastructural examination in the diagnostic evaluation of patients with suspected LSD. Biopsy of skin and conjunctiva are easily obtained and provide multiple cell types including endothelium, epithelium, fibroblasts and nerves for ultrastructural study. Fibroblasts from skin and conjunctiva can also be utilized for the initiation of tissue cultures for chemical assays. Brain biopsy has been largely replaced by biopsy of more readily obtained tissue and by biochemical assays. Such assays though may give equivical or nondiagnostic results and in some lysosomal storage diseases an enzyme defect has not yet been identified and diagnoses can be made only by ultrastructural examination.

scholarly journals Sphingolipid lysosomal storage diseases: from bench to bedside

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Muna Abed Rabbo ◽  
Yara Khodour ◽  
Laurie S. Kaguni ◽  
Johnny Stiban
Keyword(s):  
Basic Research ◽  
Lysosomal Storage Diseases ◽  
Therapeutic Strategies ◽  
Clinical Applications ◽  
Late Nineteenth Century ◽  
Lysosomal Storage ◽  
Storage Diseases ◽  
The Past ◽  
Health And Disease ◽  
General Aspects

AbstractJohann Ludwig Wilhelm Thudicum described sphingolipids (SLs) in the late nineteenth century, but it was only in the past fifty years that SL research surged in importance and applicability. Currently, sphingolipids and their metabolism are hotly debated topics in various biochemical fields. Similar to other macromolecular reactions, SL metabolism has important implications in health and disease in most cells. A plethora of SL-related genetic ailments has been described. Defects in SL catabolism can cause the accumulation of SLs, leading to many types of lysosomal storage diseases (LSDs) collectively called sphingolipidoses. These diseases mainly impact the neuronal and immune systems, but other systems can be affected as well. This review aims to present a comprehensive, up-to-date picture of the rapidly growing field of sphingolipid LSDs, their etiology, pathology, and potential therapeutic strategies. We first describe LSDs biochemically and briefly discuss their catabolism, followed by general aspects of the major diseases such as Gaucher, Krabbe, Fabry, and Farber among others. We conclude with an overview of the available and potential future therapies for many of the diseases. We strive to present the most important and recent findings from basic research and clinical applications, and to provide a valuable source for understanding these disorders.

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